Comparative Biochemistry and Physiology Part C 136 (2003) 127–134 The heart of Daphnia magna: effects of four cardioactive drugs Arturo Villegas-Navarroa, *, Esperanza Rosas-Lab , Jose´ L. Reyes aLaboratorio de Investigacion´ Ambiental, 95 Oriente No. 1649, Col. Granjas de San Isidro, Puebla Pue C.P. 72590, Mexico bPhysiology and Biophysics Department, Centro de Investigacion´ y Estudios Avanzados, IPN, Mexico DF, Mexico Received 15 May 2003; received in revised form 29 July 2003; accepted 30 July 2003 Abstract We used Daphnia magna bioassays to determine the LC50 and the effects on the heart of the cardioactive drugs ouabain, verapamil, metaproterenol and metoprolol. Distinctions were made between the pharmacological and toxicolog- ical effects of these drugs and the adequacy of physicochemical characteristics of its habitat (reconstituted water). Video microscopy and digital image processing were used to study the pharmacological effects on the heart. D. magna exhibited the expected sensitivity to the reference toxicant sodium dodecyl sulfate with a LC50 of 15.6"4.5 mgyl. All drugs were toxic with 48 h-LC50 of 2.03 mgyl ouabain, 7.04 mgyl verapamil, 32.45 mgyl metaproterenol and 76.21 mgyl metoprolol. Ouabain was the most toxic and caused a positive concentration-dependent inotropic effect. Verapamil caused positive chronotropic and inotropic effects, while metaproterenol showed positive concentration-dependent chronotropic effects at high concentrations (10y3 and 10y4 M). Metoprolol induced a positive chronotropic effect at low concentrations (10y8 , 10y7 , 10y6 M) and a negative chronotropic effect at high concentration (10y4 M). Ouabain, metaproterenol and metoprolol in D. magna caused similar effects to those produced in mammals. In contrast, verapamil caused opposite effects. The results suggest the presence of Naqq , K -ATPase receptors to verapamil and of non-specific adrenergic receptors in heart of D. magna. ᮊ 2003 Elsevier Inc. All rights reserved. Keywords: Contraction strength; Daphnia magna; Heart rate; Metaproterenol; Metoprolol; Ouabain; Verapamil 1. Introduction models has been increased (Ekwall, 1999; Keddy et al., 1995). Pharmacology has made tremendous strides in One kind of in vitro assay is the use of inver- the sophistication of the models used to identify tebrates (Yeoman and Faragher, 2001). These and understand the mechanisms of action of agents organisms have less developed cortical and sen- and in diversification of specific models to study sorial systems and are characterized by fast tissue one determined pharmacological effect (Van der regeneration. Daphnia magna has been used to Kloot, 1967; Clemedson and Ekwall, 1999; Guil- study drugs with cardiac action such as acetylcho- hermino et al., 2000). Furthermore, for reasons of line, tetraethylpyrophosphate, pilocarpine, adrena- concern about animal welfare, economics and the line and rotenone. They caused negative need for greater sensitivity and further understand- chronotropic effects on D. magna’s heart, while ing of pharmacodynamics, the interest in in vitro atropine had a positive chronotropic action and q adrenaline accelerated cardiac rate only at high *Corresponding author. Tel.yfax: 52-222-2-45-77-40. ( ) E-mail address: [email protected] concentrations Bekker and Krijgsman, 1951 . (A. Villegas-Navarro). Crozier et al. (1999) reported that nicotine slowed 1532-0456/03/$- see front matter ᮊ 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1532-0456(03)00172-8 128 A. Villegas-Navarro et al. / Comparative Biochemistry and Physiology Part C 136 (2003) 127–134 D. magna’s heart rate, while ethanol raised it. kept at 4 8C. Before use, the pellets were diluted Postmes et al. (1989) reported that agonists and in 50 ml of reconstituted water (Villegas-Navarro antagonists were either inactive or lowered the et al., 1997). heart frequency and the negative chronotropic effect of epinephrine could not be blocked by the 2.1. Forty eight hours-LC50 determination antagonist propranolol. D. magna bioassay is recommended as advan- Ten neonates were placed in each 150 ml con- tageous over other models for assessment of aquat- tainers containing 100 ml of the drug solution ic toxicity (Environment Canada, 1990) but it dissolved in reconstituted water. All assays and a remains to be shown if bioassays with D. magna control group were made in triplicate, except for are feasible and show advantages in pharmacolog- the sodium dodecyl sulfate (SDS, Sigma, St. Lou- ical studies. One advantage that can be pointed is, MO) tests, which were done in duplicate. A out is that D. magna are transparent (Chapman, 24-h preliminary test was carried out to determine 1976). Transparency is a useful characteristic to if the drug produced any effect on the physico- obtain insights into animal physiology, as it allows chemical characteristics of the solution and to the researcher to apply optical methods to visualize determine the concentration range to be used in physiological functions and to measure several the definitive test. The criterion for a valid bioas- different parameters simultaneously (Rudiger¨ et say was an unmoving rate of less than 10% in the al., 1997) and non-invasive method in physiolog- control group. In the definitive test, the minimum ical research (Colmorgen et al., 1995). number of dilutions was five plus the control The purpose of the present study is to evaluate group. Immobile organisms were counted after 48 the D. magna model to study drugs with action h to calculate 48 h-LC50 . upon the heart, and was chosen because of its size and because this organ can be easily observed by 2.2. SDS assay optical methodologies. SDS was used to determine the sensitivity of 2. Materials and methods the D. magna to this chemical according to rec- ommended procedures (Lewis and Horning, 1991). D. magna was cultured for several generations The LC50 for SDS was established in a 48 h in hard-reconstituted water as previously described bioassay in advance to the pharmacological bio- (Villegas-Navarro et al., 1997). The quality criteria assays. The concentrations were 4, 8, 16, 32 and applied to the culture of D. magna were those 64 mgyl. published by Poirier et al. (1988). For the toxicity tests, neonates of the 2nd–6th generations with 2.3. Drugs less 24 h old were used. Hard-reconstituted water for the cultures was prepared in agreement to The pharmacological solutions used were: Oua- Mexican Official Norm NMX-AA-087 (SCFI, bain, (")-verapamil hydrochloride, metaproteren- ) ( ) (q) 1995 and was aerated for 48 h to obtain O2 ol-hemisulfate and " -metoprolol -tartrate concentrations exceeding 3 mgyl (Conductronic (SIGMA, St. Louis, MO). They were selected by Model Ox25). The reconstituted water had the the following criteria: (1) by their site of action; following physicochemical parameters: pH 7.5– (2) they are prototypes of their action mechanisms; 8.5 (Corning Model 7), hardness of 160–180 mgy (3) they are used in clinical practice; and (4) they ( ) l expressed as CaCO3 Fritz and Schenk, 1969 , are soluble in reconstituted water. Before perform- conductivity of 250–600 mSycm (YSI Model ing bioassays the basic physicochemical parame- 520A) and temperature 20"3 8C. These are ade- ters of the drug solutions were measured to assess quate conditions for the growth of this species. D. that D. magna were in a satisfactory habitat to magna were set in the container for reproduction study their normal physiological function and to and growth along with 1000 ml of reconstituted discriminate between any abnormal function due water, which was changed once a week. Chlorella to physicochemical parameters and the pharmaco- vulgaris was used to feed the D. magna (Naylor logical effect. The drug solutions were placed et al., 1993) and was cultivated according to Stein under experimental conditions of light and air (1973), then concentrated and the pellets were environmental. A. Villegas-Navarro et al. / Comparative Biochemistry and Physiology Part C 136 (2003) 127–134 129 2.4. Experimental set-up ed using a 10 mm rule on the microscope stage and storing that image as a file in the computer. The main component was an inverted micro- One frame was selected with Asymetrix Software scope (IROSCOPE Model SI-PH), a digital video and transported to Corel Draw up to subfile Photo camera (PANASONIC, Model GP-KR222), a vid- Paint and used to measure areas. In Photo Paint eotape recorder VHS (SONY Model SLV-LX7S) the file-ruling image was compared to Photo Paint and an assembled computer. Image information scale throughout superimposing their images (10 was stored using VHS videotape recorders and mms9.25 mm) and heart area was estimated as a was displayed on a monitor. One recorder stored ellipse. In this way readings can be made more the original video information from the camera, conveniently and with greater accuracy. while the other stored the processed data in the computer. The software Asymetrix and Corel Draw 2.8. Statistics in a PC slot were used for on-line image processing. All data were assessed for homogeneity of variance to ensure that assumptions of analysis of 2.5. Recording of cardiac events variance ANOVA were met. The data were ana- lyzed by one-way ANOVA and the Tukey’s test to D. magna of 10 days old were used. If most of narrow down which columns were significantly the water is withdrawn, the animals are maintained different from other columns (GraphPad InStat, in place by the surface tension of the remaining Windows 95). For 48 h-LC50 the Probit method fluid and unable to displace out of the micro- (Fevrier, 1987) was used. The significance level scope’s field. Then, the frequency and the type of was set at P-0.05s*; PF0.01s**; PF0.001s muscular contraction can be observed; the area of ***. The graphic results are shown normalized: the heart in systole and diastole served to judge decrease of the areas1yvycv; dispersion values the magnitude of contraction.